Microtubules play essential roles in a variety of cellular processes, including chromosome segregation and organelle transport. They are dynamic polymers that alternate frequently between phases of growing and shrinking, a property that allows the rapid rearrangement of microtubule arrays in response to cellular signals. Microtubule dynamics are regulated, in large part, by a group of proteins referred to as plus-end tracking proteins or +TIPs. Currently, about a dozen conserved +TIP families have been identified in eukaryotes from yeast to human, but the mechanisms by which most of these proteins influence microtubule dynamics are not well understood. In addition to their ability to bind microtubules, most +TIPs interact with several other +TIPs, creating a complex web of +TIP interactions. These interactions are likely to be key regulators of +TIP activities and are the primary focus of this application. We propose to study four +TIPs that play prominent roles in regulating microtubule dynamics in the yeast, S. cerevisiae. These proteins, Bim1, Bik1, Stu1 and Stu2 belong to the +TIP families EB, CLIP, CLASP, and XMAP215, respectively. Our goal is to purify these proteins and determine how they influence microtubule dynamics in an in vitro microtubule assembly system. Then we will combine pairs of proteins to examine how particular +TIP interactions affect their abilities to regulate microtubule dynamics. We have developed all of the necessary assays in preliminary studies with Bim1 and Bik1. We now plan to expand these studies by including Stu1 and Stu2.